Glycerol is used as an intermediate for various industrial products such as cosmetics, liquid soaps, foods, medicines, and lubricants, and as a material for the fermentation industry. For example, 1,3-propanediol is produced by fermentation of glycerol.
Glycerol can be produced by fermentation, chemical synthesis, or lipolysis. With respect to glycerol production by microorganism fermentation, yeasts such as S. cerevisiae, C. magnoliae, P. farinose, and C. glycerinogenes, bacteria such as B. subtilis, and algae such as D. tertiolecta are known as a glycerol-producing microorganism.
It is known that a recombinant microorganism developed by manipulating a known glycerol biosynthetic pathway can be used as a glycerol-producing microorganism.
Generally, a carbon substrate such as glucose is converted to glucose-6-phosphate by hexokinase in the presence of ATP. Glucose-6-phosphate is converted to fructose-6-phosphate by glucose-phosphate isomerase, which is then converted to fructose-1,6-diphosphate by 6-phosphofructokinase. Fructose-1,6-diphosphate is converted to dihydroxyacetone phosphate (DHAP) by aldolase. Finally, DHAP is converted to glycerol-3-phosphate (G3P) by NADH-dependent glycerol-3-phosphate dehydrogenase (G3PDH), which is then dephosphorylated to glycerol by glycerol-3-phosphate phosphatase (Agarwal (1990), Adv. Biochem. Engrg. 41:114).
In addition, an alternative pathway for glycerol production from DHAP has been suggested (Wang et al., 1994 J. Bact. 176: 7091-7095). According to this alternative pathway for glycerol production, DHAP is dephosphorylated to dihydroxyacetone by specific or nonspecific phosphatase, which is then reduced to glycerol by dihydroxyacetone reductase. Dihydroxyacetone reductase is found in prokaryotes and Schizosaccharomyces pombe. Another alternative pathway for glycerol production from DHAP has been suggested (Redkar, Experimental Mycology, 19: 241, 1995). According to this alternative pathway for glycerol production, DHAP is isomerized to glyceraldehyde-3-phosphate by triose 3-phosphate isomerase which is a common glycolysis enzyme. Glyceraldehyde-3-phosphate is dephosphorylated to glyceraldehyde, which is then reduced by alcohol dehydrogenase or NADPH-dependent glycerol dehydrogenase.
Among genes that participate in a known glycerol biosynthetic pathway, DAR1 and GPD1 from S. cerevisiae is known as a gene encoding G3PDH for conversion of DHAP to G3P. GPP2 from S. cerevisiae is known as a gene encoding glycerol-3-phosphate phosphatase for conversion of G3P to glycerol.
In addition, there is known a method for producing glycerol using a recombinant host cell obtained by introducing a foreign gene involved in glycerol synthesis into a host cell dependent on a natural glycerol synthetic pathway. For example, U.S. Pat. No. 6,358,716 discloses a method for producing glycerol from a recombinant microorganism, which includes: (i) transforming a suitable host cell with an expression cassette including (a) a gene encoding NADH-dependent glycerol-3-phosphate dehydrogenase or NADPH-dependent glycerol-3-phosphate dehydrogenase; and (b) a gene encoding glycerol-3-phosphate phosphatase (EC 3.1.3.21); (ii) culturing the transformed host cell of (i) in the presence of at least one carbon source selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, and single-carbon substrates, to thereby produce glycerol; and (iii) recovering the glycerol produced in (ii).